Int..I. lIev. Riol. 33: 445-453 (1989) 445 Original Article Initial GABAergic expression in embryonic amphibian neuroblasts after neural induction FABIENNE PITUELLO'. PAULETTE KAN', MICHEL GEFFARD', ANNE-MARIE DUPRAT'* !URA-CNRS 675 ~ affi/hie a I'/NSERM, Centre de 8iologie du Devefoppement, Universite Paul Sabatier, Toulouse and l/nstitut de 8iologie Cellula ire et Neurochimie du CNRS, Laboratoire d'immunologie, Bordeaux, France ABSTRACT At the late gastrula-early neurula stage some embryonic neuroblasts from neural plate and neural fold present apparently as a consequence of neural induction, the capability to develop in vitro into different neuronal subpopulations (cholinergic, dopami- nergic, noradrenergic, somatostatinergic and some other peptidergic subpopulationsJ without ongoing influences from thechordamesoderm (Duprat et al., 1987). Using the same in vitro model system, the aim of the present work was to delineate the abilities of these neuroblasts to develop GABAergic traits. The initial appearance and development of GA- BAergic phenotype has been quantitated by assaying the activity of glutamic acid decar- boxylase (GAD). GAD activity was undetectable at the early gastrula stage (stage 8a) and was slightly measurable at the early neurula stage (stage 14- onset of the culture). It increased subsequently over the next 14 days in vitro. The temporal pattern of appearance and development of GAD activity in culture was in agreement with that observed in vivo. Immunocytochemical studies showed that GABA-like immunoreactivity was expressed in vitro in a subpopulation of neurons. Thus the developmental program for GAD expression and GABA phenotype maturation is acquired at least in some neuronal precursors. These data together with previously reported results on the expression of cholinergic, catechol a- minergic and peptidergic phenotypes demonstrate that different neuronal subpopulations emerge near the end of gastrulation i.e. immediately after neural induction. The embryonic origin of this neuroblast heterogeneity remains to be determined. KEY WORDS: neural induelirm, nl'llro~nu'si.\'. GARA, GAD, neural delerminalion IntrQduction mature nervous system. It was of interest to study whether NP and NF cells that arise as a direct result of neural In the gastrulated amphibian embryo, two embryonic induction have the ability to develop mature neuronal territories appear as an immediate consequence of neural phenotypes. induction; these are the neural plate (NP) and the neural Amphibian late gastrulae constitute a useful embryonic fold (NF), which are at the origin of the central and the system for this type of experimental approach. In order to peripheral nervous systems, respectively. analyze their neuronal potentialities, we isolated NP and It is now well established that the definitive develop- NF from their embryonic environment immediately after ment of derivatives of the neurectoderm is greatly influ- neural induction, dissociated them and cultured the cells in enced by the cellular environment encountered during a defined medium. We have already demonstrated that their ontogenesis. This has been particularly well docu- neuron-like cells differentiate in vitro under such condi- mented for cells originating from the NF (cf. Le Douarin, tions (Duprat et al., 19841 and that they exhibit characteris- 1982, 1986; Landis and Keefe, 1983; Coulombe and Bron- ner-Fraser, 1986; Bronner-Fraser and Fraser, 1988; Schot- zinger and Landis, 1988). However, until recently, it was AhI'rf'I,jat;o1l.1 1/s,d ;1/ this pap..,; .c\ET, 2-amino ethylisothiollrollium; BSA. bmin serum alhumin; C\f, chordamesoderm; FITC, fluoH'sn'in isothiocya- not clear to what extent the cells that have just undergone naIl'; (~:\H:\, gallllll;\-ollnino buryric acid; C.-\D, gluta1l1ic ;lei,] decarboxylase; neural induction possess the potentialities for differentia- ;\IB~', l\'a lIletabimlfite; ~-C.A.\l, neural cell adlwsiOJlll1olt'cule; :\'F, neural tion into the various phenotypes that characterize the rold; :\'P, neural plate; SE;\l, standard error (Jf tht' mean. "Address for reprints: URA-CNRS 675 - affiliee 11I'INSERM, Centre de Biologie du Developpement, Universite P. Sabatier, 118, route de Narbonne, 31062 Toulouse Cedex, FRANCE. 0214-6282/89 e CHCPreH Primed in Spain 446 F. Pilliello ct al. tic traits, such as neurofilament proteins and tetanus toxin binding sites or N-CAM (Duprat etal., 1986; Saint-Jean net et al., 1989). With regard to expression of neurotransmit- ; ters, we have provided evidence that the developmental ,i program forthe cholinergic phenotype is determined early (Duprat et al., 1984). Absent at the late gastrula stage, choline acetyltransferase and acetylcholinesterase mole- cular isoforms appear in vitro and differences in the pat- tern of expression of these enzymes were observed be- '.~.'/. !!:;. tween NP- and NF-derived neurons (Duprat et al., 1985a, 1985b). Catecholaminergic and peptidergic traits were also de- monstrated in culture of NP and NF cells isolated in vitro immediately after induction (Pituello et a/., 1989). With respect to the study of early events in neurogene- sis, we thought that it would be of interest to determine whether neuronal precursor cells acquire early the ability to express the GABAergic phenotype. GABA (gamma-amino butyric acid) is considered to be a transmitter as well as a modulator of neuronal activity (Lundberg et al., 1983; Hokfelt et al., 1984; Freund and Antal, 1988). It is assumed to be a major inhibitory trans- mitter in the central and peripheral nervous systems and GABAergic neurons are believed to be among the main mediators of recurrent inhibition, playing a crucial role in the selection of responses to different stimuli. This regula- tory molecule has been implicated in the pathophysiology NP of a number of neurological and psychiatric disorders. Nf Differentiation of cells displaying a GABAergic pheno- type can be conveniently monitored by measurement of the activity of glutamic acid decarboxylase (GAD), the enzyme catalyzing the synthesis of GABA and/or by immu- NP nocytochemical detection of the amino acid itself. The aims of the present study were to use NP and NF ~,~f ~ cells isolated in vitro straight after neural induction (Du- CM prat et al., 1984, 1985a, 1985bl in order to: (i) determine the ability of these embryonic cells to develop GABA-related properties (GAD activity, GABA biosynthesis, accumulation and uptake); (ii) study the temporal pattern with which the GABAer- gic phenotype emerges and matures in differentiating neu- rons from NP and from NF at the late gastrula stage, in vitro and in vivo; Wi) visualize immunocytochemically and quantify the GABAergic cell population differentiating in vitro. Results Biochemical studies of initial expression of GAD activity and changes in the kinetics of expression during development in vitro and in vivo Cell cultures Fig. 1. Experimental procedure for cell cultures. Five types of cultures The appearance and the development of GAD, the en- were performed from late gastrula embryos (stage 13): (l)Cocultured zyme responsible for GABA biosynthesis, were analyzed neurecrodermal cells mixed with underlying chordamesodermal cells. i.e.. quantitatively in cocultures (NP + NF + CM), and in NP and coculrures (NP + NF + CM). (II) Isolated neural plate cells (NPl. (III) Isolated neural fold cells (NF). (IV) Isola red chordamesodermal cells (CM). (V) NF cultures. The data are schematized in Fig. 2. GAD Neurectodermal cells (NP + NF) cultured without CM. All the cultures were activity, undetectable at the early gastrula stage (stage 8a, In Barth's defined saline medium. data not shown), was very low at the late gastrula-early Illitial GABAergic expressioll 447 GABA fmol./min.lexplant 40 30 20 Fig. 2. Appearance and development of GAD activity in cell cultures from late 10 gastrulae. Analyses were performed at the onset of the culture (r = 0) and after 4-, 7- and 14 days in culture. GAD activity was measured in 4 sibling cocultures fNP + NF + CM). NP cultures and NF cultures in two sets of exper- 0 " iments fcf. Materials and Methods). GAD acti- 0 4 7 14 days vity is normalized to 'explant" which is one embryo equivalent. The data are means:!::SEM. Th corresponds to the theoretical value ob- IIIIIIII Coculture IIIIIIII NP ~NF Dth tained by addition of NP + NF values. neurula stage in cocultures assayed at the beginning of the increase in the number of positive neurons. Immunocyto- culture period. The level oftha enzyme rose regularly from chemical visualization and quantification of the GABAergic then on and, after 14 days of culture, had increased approx~ population will provide an answer to the question. imately 35 fold. In NP cultures, GAD activity was clearly demonstrable at In vivo emergence and development 4 days and subsequently increased. However, the level of GAD activity was assayed in whole embryos at a number enzymatic activity was always lower than in cocultures of of stages, four of which corresponded to time points at identical age. which cultures had been assayed (see legend to Fig. 4). In NF cultures, GAD activity was also expressed at day 4 As mentioned previously, no GAD activity was detected and increased throughout the culture period. At all time before gastrulation. A barely significant activity (O.65:tO.35 points, the enzyme levels were lower than in the corre- fmol/min/embryo) was measured at stage 13-14 (corre- sponding NP cultures. sponding to zero time of culture). Thereafter the activity A two-way analysis of variance